The generation of meaningful behavioral responses to visual stimuli requires the appropriate topographic ordering of synaptic connections throughout the visual pathway. Visual information is routed through the axons of retinal ganglion cells directly from the retina to the superior colliculus (SC) of rats, or its homologue in non-mammalian vertebrates, the optic tectum. Our studies are directed toward defining the mechanisms involved in establishing topographic connections in retinal connections using the rat SC and the chick tectum as models. Our work in the previous funding period suggested that position- encoding molecules are present in the SC as developing retinal axons enter it, but that these molecules fail to guide or restrict the growth of retinal axons to the topographically correct part of the SC. These findings suggest a fundamentally different mechanism for the development of topographic maps in this system; specifically that position-encoding molecules may control the development of topographic maps not by directing the growth of primary retinal axons, but by controlling the formation of collateral branches which go on to develop topographically ordered synaptic connections. The first two aims in this proposal extend these previous studies by using time-lapse video imaging to provide definitive evidence for mechanisms of collateral branching and arborization of retinal axons in situ in the developing SC and in vitro in a controlled environment. The development of retinotopic maps is widely believed to be controlled by targeting molecules present in a regional or graded distribution in the SC/tectum. Recent evidence has correlated the expression of En, the vertebrate homologue of the Drosophila engrailed gene, a homeobox transcription factor, with the polarity of the retinotectal map and the gradient of laminar differentiation in the tectum. The last three aims use recombinant retroviruses to over-express En-1 and En-2 in the developing tectum to determine their role in establishing tectal polarity. The first specific aims of this proposal are: (1) To analyze with time- lapse imaging the dynamics, topographic specificity and significance of retinal axon branching in the developing SC; (2) To assess in vitro using time-lapse video imaging potential mechanisms for generating topographic specificity in retinal axon branching; (3) To determine if En regulates the rostral-caudal axis of the retinotectal map; (4) To determine if En regulates the expression of retinal targeting molecules in the optic tectum; and (5) To determine if En regulates the histogenetic gradients of the optic tectum.